D-arabinitol dehydrogenase from Candida tropicalis ATCC 750 or Candida shehatae

ABSTRACT

A D-arabinitol dehydrogenase enzyme is disclosed. The enzyme is capable of catalyzing the oxidation of D-arabinitol and substantially incapable of catalyzing the oxidation of D-mannitol and is substantially free of other enzymes capable of oxidizing D-mannitol. Also disclosed are methods for determining D-arabinitol. In one embodiment the method comprises the steps of providing in combination (1) a medium suspected of containing D-arabinitol and (2) a D-arabinitol dehydrogenase enzyme and examining the medium for the product of the oxidation of the D-arabinitol. The enzyme utilized is capable of catalyzing the oxidation of D-arabinitol and substantially incapable of catalyzing the oxidation of D-mannitol. Kits for conducting the present method are also disclosed. The D-arabinitol dehydrogenase in purified form has all of the characteristics of a D-arabinitol dehydrogenase which is obtainable from Candida tropicalis ATCC 750 or Candida shehatae.

This application is a continuation of U.S. Ser. No. 08/184,764 filedJan. 21, 1994, now abandoned, which is a continuation U.S. Ser. No.07/731,218, filed Jul. 12, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

At present the laboratory tools available to clinicians for thediagnosis of invasive candidiasis are limited. Surveillance cultures ofperipheral sites have little predictive value, with the possibleexception of Candida tropicalis infection in neutropenic patients. Bloodcultures, even when they are tailored for optimal growth of fungi, areslow, insensitive, and nonspecific. Measurement of antibodies to Candidais not helpful in immunosuppressed patients who comprise the very groupthat is most vulnerable to invasive infections. Detection of circulatingfungal products, particularly mannan, provides the desired level ofspecificity, but the clinical sensitivity of current assays isdisappointing, and the technology for performing them is not readilyportable to clinical laboratories. Commercially available latexagglutination kits detect uncharacterized antigen(s) and lacksensitivity and specificity.

Most medically important species of Candida produce micromolar amountsof the pentitol D-arabinitol in vitro and there is considerable evidencethat patients with invasive candidiasis have higher serum D-arabinitollevels and higher serum D-arabinitol/creatinine ratios than uninfectedpatients. Therefore, D-arabinitol is potentially useful as a diagnosticmarker for invasive candidiasis, a disease that often is difficult todiagnose antemortem by traditional methods.

Enantioselective measurements of D-arabinitol in human serum wereoriginally made by combined microbiologic-gas chromatographic (GC) andenzymatic-GC techniques. These approaches were, however, time consumingand cumbersome. Recently, two GC methods have been developed that employcolumns with a chiral stationary phase capable of separating enantiomersof arabinitol. Although these methods are highly specific forD-arabinitol and do not require serum pretreatment by enzymatic ormicrobiologic techniques, they are too cumbersome for routine use in theclinical laboratory. A more practical enzymatic fluorometric method hasbeen developed that uses an Enterobacter aerogenes (Klebsiellapneumoniae) D-arabinitol dehydrogenase. Unfortunately, cross reactivityof the dehydrogenase with D-mannitol, a hexitol normally present inhuman serum, reduces the specificity of this assay.

2. Description of the Related Art.

Soyama and Ono, in Clinica Chimica Acta, 149 (1985) 149-154, describe anenzymatic fluorometric method for the determination of D-arabinitol inserum by initial rate analysis.

Soyama and Ono describe an improved procedure for determining serumD-arabinitol by a resazurin-coupled enzymatic method in Clinica ChimicaActa, 168 (1987) 259-260.

The purification and properties of Klebsiella aerogenes D-arabinitoldehydrogenase are discussed by Neuberger, et al., in Biochem. J., 183(1979) 31-42.

Kiehn, et al., in Science, 206 (1979) 577-580, describe a gas-liquidchromatographic method for measuring D-arabinitol in human serum andassess the clinical usefulness of this method for detecting candidiasis.In actuality, total pentitol (D- and L-arabinitol, xylitol and ribitol)concentration is being measured by this method.

Bernard, et al., in J. Infect. Dis., 151(4)(1985) 711-715, describe acombined microbiologic-GC method for determining the stereoisomericconfiguration of arabinitol in serum, urine, and tissues in invasivecandidiasis. In this method, D-arabinitol concentrations are calculatedas the difference between serum arabinitol levels determined by GCbefore and after sample incubation with a strain of C. tropicalis, whichconsumes D-arabinitol once preferred substances are exhausted.Unfortunately, the method requires a 24-hour incubation step, issusceptible to interference by anti-fungal drugs, and is insufficientlysensitive.

Wong and Brauer, in J. Clin. Microbiol. 26 (1988) 1670-1674, describe acombined enzymatic-GC method for the enantioselective measurement ofD-arabinitol in human serum. In this method, D-arabinitol dehydrogenasefrom K. pneumoniae is used instead of C. tropicalis cells for theremoval of D-arabinitol from serum; and D-arabinitol levels arecalculated as the difference between arabinitol levels determined by GCin the untreated and enzyme-treated serum. Although this combinedenzymatic-GC method is unaffected by antifungal drugs, sufficientlysensitive to quantify D-arabinitol in most serum specimens, and can becompleted within a few hours, the technique requires that each specimenbe analyzed twice by GC to determine the concentration of D-arabinitol.

Enantioselective measurement of the Candida metabolite D-arabinitol inhuman serum using multidimensional gas chromatography and a new chiralstationary phase is disclosed by Wong and Castellanos, in J.Chromatography, 495 (1989) 21-30. This technique is sensitive and highlyspecific for D-arabinitol, but requires that each specimen be fractionedsuccessively over GC columns containing a conventional and chiralstationary phase.

The separation and quantification by gas chromatography-massspectrometry of arabinitol enantiomers to aid the differential diagnosisof disseminated candidiasis is disclosed by Roboz, et al., in J.chromatog., 500 (1990) 413-426. The columns used in this approach have alimited useful lifetime and the procedure is laborious andtime-consuming.

A review of techniques for the diagnosis of invasive candidiasis isdescribed by Jones in Clin. Microbiol. Rev., 3 (1990) 32-45.

Ness, et al., in The Journal of Infectious Diseases, 159 (1989) 495-502describe the Candida antigen latex test for detection of invasivecandidiasis in immunocompromised patients.

Cabezudo, et al., discuss the value of the Cand-Tec Candida antigenassay in the diagnosis and therapy of systemic candidiasis in high-riskpatients (Eur. J. Clin. Microbiol. Infect. Dis., 8 (1989) 770-777).

Walsh, et al., in N. Engl. J. Med., 324(15) (1991) 1026-1031, assess theclinical utility of the Candida enolase antigen test for detectingcandidiasis in cancer patients.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to an enzyme inpurified form specific for D-arabinitol.

Another embodiment of the present invention is directed to aD-arabinitol dehydrogenase enzyme capable of catalyzing the oxidation ofD-arabinitol and substantially incapable of catalyzing the oxidation ofD-mannitol and that is substantially free of other enzymes capable ofoxidizing D-mannitol.

Another embodiment of the present invention is a method for determiningD-arabinitol. The method comprises the steps of providing in combination(1) a medium suspected of containing D-arabinitol and (2) a D-arabinitoldehydrogenase enzyme and examining the medium for the product producedas a result of the oxidation of the D-arabinitol. The enzyme utilized iscapable of catalyzing the oxidation of D-arabinitol and substantiallyincapable of catalyzing the oxidation of D-mannitol.

Another aspect of the invention is a method for detecting the presenceof a Candida organism in a host. The method comprises the step ofexamining a sample from the host for the presence of D-arabinitolutilizing a D-arabinitol dehydrogenase enzyme. The enzyme is capable ofutilizing D-arabinitol as a substrate and substantially incapable ofutilizing D-mannitol as a substrate.

Another embodiment of the invention concerns a method of detecting aCandida infection in a patient. The method comprises the steps ofproviding in combination a sample from the patient, a D-arabinitoldehydrogenase enzyme capable of catalyzing the oxidation of D-arabinitolby nicotinamide adenine dinucleotide (NAD⁺) and substantially incapableof catalyzing the oxidation of D-mannitol, and examining the combinationfor the amount of NADH formed in a given time. The term "NAD^(+") shallmean the natural coenzyme and NAD⁺ mimetics including NAD⁺ attached to asupport and thio analogs, which contain one or more sulfur atoms inplace of one or more oxygen atoms of the NAD⁺. The amount of NAD⁺reduced form (NADH) is used to assist in the clinical diagnosis of aCandida infection.

Another aspect of the present invention involves a compositioncomprising (1) a D-arabinitol dehydrogenase enzyme capable of utilizingD-arabinitol as a substrate and substantially incapable of utilizingD-mannitol as a substrate and (2) NAD⁺.

Another embodiment of the present invention concerns a D-arabinitoldehydrogenase enzyme capable of binding to at least one of themonoclonal antibodies selected from the group consisting of 3D6 and5E11, where the enzyme is substantially free of other enzymes capable ofoxidizing D-mannitol.

Another embodiment of the present invention concerns a compositioncomprising an enzyme where the composition is capable of catalyzing theoxidation of D-arabinitol at a rate at least 20-fold faster than itcatalyzes the oxidation of any other naturally occurring polyol.

Another embodiment of the invention is a kit comprising in packagedcombination (1) a D-arabinitol dehydrogenase enzyme preparationsubstantially incapable of catalyzing the oxidation of D-mannitol and(2) NAD⁺.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention is directed to an enzymatic D-arabinitol assayutilizing a particular D-arabinitol dehydrogenase enzyme. The presentassay can be performed in much shorter times than known assays andpermits following the course of anti-fungal treatment of a Candidainfection. Thus, the present invention provides for the detection of aCandida infection based on the determination of D-arabinitol usually inserum or urine. The particular D-arabinitol dehydrogenase enzymeemployed permits the discrimination between D-arabinitol and othermetabolites such as D-mannitol.

The D-arabinitol dehydrogenase (DADH) enzyme employed is capable ofcatalyzing the oxidation of D-arabinitol and substantially incapable ofcatalyzing the oxidation of D-mannitol and is substantially free ofother enzymes capable of oxidizing D-mannitol. DADH is an enzyme havingthe I.U.B. classification of oxidoreductase acting on the CH-OH group ofdonors having D-arabinitol as a substrate.

The term "capable of catalyzing the oxidation of D-arabinitol" meansthat the pure DADH has a specific activity for catalysis of theoxidation of D-arabinitol of at least 50 international units (IU)/mg,preferably at least 100 IU/mg, more preferably at least 150 IU/mg.

The term "substantially incapable of catalyzing the oxidation ofD-mannitol" means that the DADH used in the assay has a specificactivity for catalysis of the oxidation of D-mannitol less than 1%,preferably less than 0.2%, more preferably less than 0.1% of thespecific activity for catalysis of the oxidation of D-arabinitol.

The term "substantially free of other enzymes capable of oxidizingD-mannitol" means that any other enzyme present as an impurity of theDADH is substantially incapable of catalyzing the oxidation ofD-mannitol.

The term "specific DADH" means DADH that is substantially incapable ofcatalyzing the oxidation of D-mannitol and is substantially free ofother enzymes capable of oxidizing D-mannitol.

The term "DADH in purified form" means a DADH having a purity of greaterthan 80%, preferably greater than 90%, more preferably greater than 95%,as determined by sodium dodecylsulfate (SDS) polyacrylamide gelelectrophoresis (PAGE). Upon purification of DADH, the DADH in purifiedform will catalyze the oxidation of other polyols commonly found inhuman serum at a rate at least 10-fold less than that for D-arabinitol,preferably at least 20-fold less.

The DADH of the present invention can be obtained in a number of ways.For example, the DADH of the invention can be isolated from certainmembers of the genus Candida. The enzyme is preferably obtained fromCandida tropicalis and Candida shehatae. DADH enzyme from other speciescan be identified by determining whether the DADH enzyme can bind to atleast one of the monoclonal antibodies to the DADH from Candidatropicalis selected from the group consisting of 3D6 and 5E11. Thesemonoclonal antibodies are prepared by standard hybrid cell technologyand are described herein.

An example, by way of illustration and not limitation, of a purificationprotocol for DADH from Candida tropicalis is as follows: The cellsutilized for obtaining the enzyme of the present invention arepreferably cultivated in a liquid nutrient medium that containsD-arabinitol as a carbon source as well as nitrogen, vitamins and tracemetals. The cells are grown to late log phase at room temperature on agyrotary shaker. The cells are then harvested, washed, pelleted andresuspended in a suitable buffer containing the appropriate proteinaseinhibitors. The cells are then disrupted. Yeast cells are usuallysubjected to mechanical breakage such as a high speed vibrating beadmill or high pressure shearing, which is accomplished with, for example,a French pressure cell. Bacterial cells may be disrupted enzymatically(e.g., lysozyme), with ultrasound, or by the two methods describedabove. The resultant cell suspension is then subjected tocentrifugation(s) that pellet unbroken cells, cell wall material andpossibly membranes and ribosomes. The supernatant is then treated withhighly positively charged polymers such as protamine sulfate, which willselectively precipitate nucleic acids and their associated proteins. Theenzyme is then precipitated from the solution with a salt (e.g.,ammonium sulfate), organic solvent (e.g., acetone), or organic polymer(e.g., polyethylene glycol). Final purification of the enzyme may becarried out using standard techniques such as ion exchangechromatography, reverse-phase chromatography, gel exclusionchromatography, gel electrophoresis, isoelectricfocusing, immunoaffinitychromatography, dye ligand chromatography, and the like.

An enzyme in accordance with the present invention can also be preparedby recombinant DNA technology. Briefly, a gene coding for a DADH enzymeof the invention is obtained, usually by isolation from genetic matterof the organism from which the enzyme was isolated. Generally, the geneis isolated by partial digestion of the DNA followed by centrifugationthrough a gradient material such as sucrose. Gene fragments are clonedinto a suitable cloning vector such as, for example, a plasmid, which istransfected into a host such as, for example, a bacterium, e.g.,Escherichia coli.

Alternatively, the vector may be other than a plasmid, for example,bacteriophage or cosmid. The particular vector chosen should becompatible with the contemplated host, whether a bacterium such as E.coli, yeast, or other cell. The plasmid should have the proper origin ofreplication for the particular host cell to be employed. Also, theplasmid should impart a phenotypic property that will enable thetransformed host cells to be readily identified from cells that do notundergo transformation. Such phenotypic characteristics can includegenes providing resistance to growth inhibiting substances, such as anantibiotic. Plasmids are commercially available that encode proteinsresponsible for resistance to antibiotics, including tetracycline,streptomycin, penicillin and ampicillin. Also required of plasmidvectors are suitable restriction sites that allow the ligation offoreign genes. Similar characteristics will be considered for choosingvectors other than plasmids.

The host cells carrying the gene are selected and gene expression ismonitored. If expression is low, the synthesis of DADH in the bacteriamay be improved by providing an inducible promoter at the 5'-end of thegene. Such promoters are found in commercially available expressionplasmids. Once the gene has been expressed to appropriate levels, theprotein is extracted from the bacterium. The DADH enzyme is separatedfrom other proteins by procedures described above. As mentioned above,one way of screening cultures to determine whether a DADH, in accordancewith the present invention, is obtained is to utilize a monoclonalantibody that specifically recognizes such DADH. Monoclonal antibodiesfor this purpose may be synthesized by standard hybrid cell technologybased on that reported by Kohler and Milstein in Nature 256(1975)495-497. Briefly, a host is immunized with the specific DADH enzymeisolated as described above from Candida tropicalis. The enzyme isinjected into the host, usually a mouse or other suitable animal, andafter a suitable period of time the spleen cells from the mouse areobtained. Alternatively, unsensitized cells from the host can beisolated and directly sensitized with the DADH enzyme isolate in vitro.Hybrid cells are formed by fusing the above cells with an appropriatemyeloma cell line and cultured. The antibodies produced by the culturedhybrid cells are screened for their binding affinity to the DADH enzyme.A number of screening techniques may be employed such as, for example,ELISA screens including forward and reverse ELISA assays. The screeningassays can be conducted utilizing DADH enzyme alone or in conjunctionwith a second enzyme such as, for example, diaphorase, which achieves areduction in background and an increase in the positive signal.

In the forward assay specific DADH enzyme is provided on a suitablesurface such as a microtiter plate. Supernatants from each of the hybridcolonies are individually applied to separate microtiter plate wells.After incubation, the wells are washed and goat antimouse antibodiescovalently linked to alkaline phosphatase are added to each of thewells. The wells are again incubated and washed and then filled with asubstrate for the phosphatase, such as, for example, p-nitrophenylphosphate. The wells are then observed for a signal, which indicates thepresence of an antibody specific for DADH. Hybrid cells so identifiedare subjected to recloning.

In the reverse assay the microtiter plate well is coated with rabbitantimouse antibodies. A supernatant from each of the hybrid cellcolonies is applied to separate wells. The wells are incubated andwashed, and a DADH enzyme preparation is added. NAD⁺ and D-arabinitolare added to the well. The wells are screened for the reduction of NAD⁺to NADH and the hybrid cells of positive wells are selected and reclonedto select for the hybrid cells that secrete a homogeneous population ofantibodies specific for DADH.

The monoclonal antibodies may also be prepared by cloning and expressingnucleotide sequences or mutagenized versions thereof coding at least forthe amino acid sequences required for specific binding of naturalantibodies.

Monoclonal antibodies may include a complete immunoglobulin, or fragmentthereof, which immunoglobulins include the various classes and isotypes,such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragmentsthereof may include Fab, Fv and F(ab')2, Fab', and the like.

As mentioned above, the DADH monoclonal antibodies prepared inaccordance with the above may be utilized in assays to screen enzymepreparations isolated from different organisms to obtain and identifythe DADH enzyme preparations of the present invention. Particularmonoclonal antibodies in accordance with the present invention are thoseselected from the group consisting of those antibodies recited in Table2 hereinbelow. Thus, another aspect of the present invention is a DADHenzyme capable of binding to one of the above group of monoclonalantibodies, where the enzyme is substantially free of other enzymescapable of oxidizing D-mannitol.

Another aspect of the present invention involves a method fordetermining D-arabinitol. The method comprises the steps of providing incombination (1) a medium suspected of containing D-arabinitol, and (2) aspecific D-arabinitol dehydrogenase enzyme. The enzyme is capable ofcatalyzing the oxidation of D-arabinitol and substantially incapable ofcatalyzing the oxidation of D-mannitol. Many of the DADH enzymes thatcan be used in the method will be capable of binding to a DADHmonoclonal antibody as described above. Generally, the binding of DADHenzyme to the monoclonal antibody will be such that the binding affinityis greater than 10⁶ M⁻¹, preferably greater than 107 M° , morepreferably greater than 10⁸ M⁻¹.

The medium suspected of containing D-arabinitol is generally an aqueousmedium which contains a sample from a host. The sample is usually a bodyfluid. Preferably, the blood of a patient is obtained and the serumfraction is isolated. Alternatively, urine can be used. After anappropriate incubation time, the medium is examined for the product ofthe oxidation of the D-arabinitol. The amount of D-arabinitol present isdetermined, usually by reference to a standard curve. The method hasapplication for the detection of the presence of a Candida organism in ahost. The presence of Candida is correlated to the amount ofD-arabinitol and is usually more closely correlated to the ratio ofD-arabinitol to creatinine in the sample. Creatinine is determined byany convenient commercially available method.

A cofactor must be included in the aqueous medium. When present inconcentrations in excess of the expected concentration of D-arabinitol,the cofactor serves as the oxidant. Alternatively, it may be present incatalytic concentration, in which case an auxiliary oxidant must bepresent such as, e.g., pyruvate and lactate dehydrogenase. Usually, thecofactor is NAD⁺ or a derivative thereof such as NADP⁺. When thecofactor is in excess of the expected concentration of D-arabinitol, themedium can be examined for the reduced form of the cofactor after thecombination is incubated. For NAD⁺ the reduced form will be NADH. Theamount of the reduced cofactor formed in a given time over apredetermined amount is indicative of the amount of D-arabinitol in thesample. The amount of reduced cofactor may be detected directly, e.g.,by measuring an amount of fluorescence, or indirectly, e.g., by addingadditional reagents.

In one embodiment of the method of the present invention the medium isexamined for the amount of the reduced cofactor by adding to the mediuma chromophoric reagent that is capable of being reduced by the reducedform of the cofactor. The chromophoric reagent can serve as an auxiliaryoxidant and may be present in the medium at the beginning of theincubation period or may be added following incubation. When addedfollowing incubation, it will not serve as an auxiliary oxidant. Whenreduced by the reduced cofactor, the chromophoric reagent will provide adetectible signal. Such chromophoric reagents include, by way of exampleand not limitation, resazurin, tetrazolium salts such asp-iodophenylnitrophenyltetrazolium, Fe^(III) -phenanthroline complex,and the like.

In the embodiment of the present invention utilizing chromophoricreagents, a catalyst that assists in the reduction of the chromophoricreagent by the reduced cofactor is added to the assay medium. Thecatalyst will usually be capable of being reduced by the reducedcofactor and the reduced form of the catalyst will usually be capable ofreducing the chromophoric reagent. Typical catalysts that are usefulinclude diaphorase, phenazine methosulfate, meldola blue,1-hydroxy-5-alkylphenazinium salts, methylene blue, etc. With NADH andeither resazurin or a tetrazolium salt, the enzyme diaphorase isemployed as a catalyst.

When a chromophoric reagent is utilized in the method of the presentinvention, the product is usually detected spectroscopically. Forexample, the measurement may be made by detection of fluorescence, lightabsorption, chemiluminescence, light scattering, electroluminescence,etc. Alternatively, the chromophoric reagent need not be converted to aconventional chromophore but is instead converted to a substance thatcan be detected by nonspectroscopic means such as electrochemicaldetection.

In a preferred embodiment of the present invention, the assay is carriedout in a two-stage incubation in the presence of an amount of NAD⁺ thatwill usually be in excess of the highest expected amount ofD-arabinitol. The oxidation of D-arabinitol is catalyzed by the DADHenzyme in the initial incubation followed by the catalyzed oxidation ofthe NADH to NAD⁺ in the presence of a chromophoric reagent. By addingthe catalyst and/or chromophoric reagent following the initialincubation, these reagents can be added in sufficient amounts to causethe reaction with the NADH to occur rapidly and completely. Inclusion ofthese reagents during the initial incubation also provides usefulresults, but conversion of the chromophoric reagent to a chromophore bysubstances in the sample other than D-arabinitol introduces a higherbackground signal and reduces assay sensitivity.

The method and compositions of the invention may be adapted to mostassay formats. The assays may be homogeneous or heterogeneous. In ahomogeneous assay approach, the sample may be pretreated if necessary toremove unwanted materials. The reaction usually involves a DADH enzymeand a sample from a patient suspected of a Candida infection. Asmentioned above, cofactors, a chromophoric reagent and a catalyst may beincluded.

The above materials are combined in an aqueous assay medium and themedium is examined for the reduced cofactor. Both the reaction anddetection of the extent thereof are carried out in a homogeneoussolution. The reaction can be carried out utilizing a one stage or twostage incubation. A two stage incubation is preferably employed. Otherapproaches for detecting this oxidation reaction are measuring the rateof incorporation of tritium into D-arabinitol when the sample, NAD and[4(S)-³ H]NADH are incubated, or the separation and chromophoricdetection of D-ribulose formed by oxidation of D-arabinitol.

In a heterogeneous assay approach, the reagents are as described abovein the homogeneous approach. Generally, the D-arabinitol will beseparated from the bulk of the sample, usually by a chromatographicmethod. The separated D-arabinitol will then be oxidized by use of aspecific DADH and an NAD derivative and the product(s) detected asalready described.

The assay will normally be carried out in an aqueous buffered medium ata moderate pH, generally that which provides optimum assay sensitivity.The assay can be performed either without separation (homogeneous) orwith prior separation (heterogeneous) of any of the assay components orproducts.

It may be desirable or preferable to pretreat a sample to be assayed toremove interferents prior to conducting an assay. The sample can besubjected to, for example, ultrafiltration or elevated temperatures toaccomplish this pretreatment. Samples include body fluids such as serum,whole blood, urine, etc.

The aqueous medium may be solely water or may include from 0.01 to 10volume percent of a cosolvent. The pH for the medium will usually be inthe range of about 5 to 11, more usually in the range of about 6 to10.5, and preferably in the range of about 7 to 10. The pH value willusually be selected to maximize the extent and rate of oxidation ofD-arabinitol without causing excessive decomposition of the DADH.Generally, the reaction will progress further toward completion as thepH is increased and as the cofactor concentration is increased.

Various buffers may be used to achieve the desired pH and maintain thepH during the determination. Illustrative buffers include glycine,phosphate, carbonate, tris, barbital and the like. The particular bufferemployed is not critical to this invention, but in an individual assayone or another buffer may be preferred. Borate is not suitable for thisassay.

Moderate temperatures are normally employed for carrying out the assayand usually constant temperatures during the period of the measurement.Incubation temperatures will normally range from about 5° to 45° C.,more usually from about 15° to 40° C. Temperatures during measurementswill generally range from about 10° to 50° C., more usually from about15° to 40° C.

For detection of Candida infection the concentration of D-arabinitolthat may be assayed will generally vary from below 1×10⁻⁷ to 1×10⁻³ M,more usually from about 1×10⁻⁶ to 5×10⁻⁴ M. In serum, the clinicallyrelevant concentration will usually range from about b 1×10⁻⁶ to 1×10⁻⁴M. Considerations such as the sensitivity of a particular detectiontechnique, the time desired to complete the assay, the cost of thereagents and the concentration of the D-arabinitol will normallydetermine the concentrations of the various reagents.

The concentration of the NAD⁺ or a derivative thereof will affect thevalue and equilibrium of the reaction. Normally, in order to minimizethe assay time, the NAD⁺ concentration should be at least equal to theK_(m) of the enzyme with respect to NAD⁺. Further increasing theconcentration of NAD⁺, for example, to 10-100 K_(m), is useful when lowconcentrations of D-arabinitol are present in order to assure maximalconversion to NADH. As for the enzyme, the greater the concentration ofthe enzyme the faster the reaction is completed. Usually it is desirableto use at least 0.1 IU, preferably at least 1 IU, more preferably atleast 10 IU. However, the cost of this reagent may dictate the use ofless than a most preferred concentration. The concentrations of reagentsused to detect the NADH that is formed will depend on the particulardetection method used, the required sensitivity of the method and thepotential for producing non-specific background signal with excessreagents.

While the order of addition may be varied widely, there may be certainpreferences. The simplest order of addition is to add all the materialssimultaneously and determine the signal produced. Alternatively, thereagents can be combined sequentially. As described above, one or moreincubation steps may be involved subsequent to each addition, eachgenerally ranging from about 10 seconds to 6 hours, more usually fromabout 30 seconds to 1 hour.

In a homogeneous assay after all of the reagents have been combinedeither simultaneously or sequentially, the presence of product producedas a result of the oxidation of D-arabinitol is determined. The presenceand amount of such product is related to the amount of the D-arabinitolin the sample tested. The DADH enzyme of the invention is preferablyfirst combined with the sample and enzyme cofactors. After incubation,either or both the chromophoric reagent and catalyst required to permitreaction of the NADH with the chromophoric reagent are added if notalready included in the assay medium. The amount of chromophoric reagentemployed is usually at least equimolar to the maximum amount ofD-arabinitol that is expected in the assay medium, preferably at least10 times the amount of D-arabinitol, more preferably at least 100 timesthe amount of D-arabinitol.

Another aspect of the present invention relates to kits useful forconveniently performing the assay method of the invention fordetermining the presence or amount of D-arabinitol in a sample suspectedof containing the D-arabinitol. To enhance the versatility of thesubject invention, the reagents can be provided in packaged combination,in the same or separate containers, so that the ratio of the reagentsprovides for substantial optimization of the method and assay. Thereagents may each be in separate containers or various reagents can becombined in one or more containers depending on the cross-reactivity andstability of the reagents. The kit comprises as one reagent a specificDADH enzyme in accordance with the invention. The kit can furthercomprise a cofactor such as NAD⁺, and an agent that reacts with NADH togive a detectable product.

The kit can further include other separately packaged reagents forconducting an assay in accordance with this invention such as supports,ancillary reagents, sample pretreatment reagents, and so forth. Asupport can be a porous or non-porous water insoluble material. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders, natural polymeric materials; synthetic ormodified naturally occurring polymers, such as plastics; glass;ceramics; metals and the like.

Another embodiment in accordance with the present invention is directedto a composition comprising NAD⁺ and a specific DADH enzyme capable ofutilizing D-arabinitol as a substrate and substantially incapable ofutilizing D-mannitol as a substrate.

Another embodiment of the present invention is a composition comprisingan enzyme which is a dehydrogenase capable of catalyzing the oxidationof D-arabinitol at a rate of at least 10-fold faster than it catalyzesthe oxidation of any other naturally occurring polyol, preferably atleast 20-fold faster.

Another embodiment of the present invention is a composition comprisinga DADH enzyme that binds to either or both of the monoclonal antibodies3D6 and 5E11 with an affinity constant of at least 1×10⁶ M⁻¹, preferablyat least 1×10⁸ M⁻¹. Another embodiment of the present invention is a C.shehatae enzyme that binds either or both of the monoclonal antibodies3D6 and 5E11. Another embodiment of the present invention is DADH fromC. tropicalis that also binds either or both of the monoclonalantibodies 3D6 and 5E11.

EXAMPLES

The invention is further demonstrated by the following illustrativeexamples. Parts and percentages used herein are by weight unlessotherwise indicated. Temperatures are in degrees centigrade (° C).

Example 1

PURIFICATION OF D-ARABINITOL DEHYDROGENASE FROM CANDIDA TROPICALIS

Centrifugation and other protein purification steps were performed at 4°unless otherwise noted. Four liters of cells were grown in YeastNitrogen Base (Difco) supplemented with 0.5% (w/v) D-arabinitol on agyrotary shaker at room temperature. The cells were harvested bycentrifugation after they reached late log phase in their growth cycle(OD₆₀₀ >5.0). The cells were then washed with distilled water,repelleted and the wet weight of the cell pellets was determined. Thepellets were resuspended in 0.1M NaH₂ PO₄, 10⁻⁷ M pepstatin A, 1 mMphenylmethylsulfonyl fluoride, pH 7.0 with NaOH at room temperature,using two mls of the above buffer per gram of wet weight cells.Acid-washed glass beads (0.45-0.55 mm) were added to the resuspendedpellets using two grams of beads for each gram of wet weight cells. Thecells were then disrupted in a Braun MSK Cell Homogenizer for 5 minwhile being cooled with liquid nitrogen. The disrupted cells wereremoved from the glass beads and were spun at 5,000×g for 5 min topellet cell wall material. The supernatant was removed and spun at100,000×g at 4° for 1 hr in an ultracentrifuge. The resultingsupernatant was removed and its volume and protein concentration wasdetermined. For every gram of protein, 90 mg of protamine sulfate wasadded dropwise from a 2% (w/v) stock over 5 min while stirring on ice.After equilibrating the solution by stirring for an additional 15 min onice, the nucleic acid-protein precipitate was removed by centrifugationat 30,000×g for 15 min. The resulting supernatant was brought to 40%saturation with ammonium sulfate crystals, which were added over aperiod of 20 min while stirring the solution on ice. After equilibratingthe solution by stirring for an additional 30 min on ice, the proteinprecipitate was recovered by spinning the solution at 100,000×g for 15min. The resulting pellet was resuspended in running buffer (50 mM NaH₂PO₄, 0.5M NaCl, 5 mM MgCl₂, 10⁻⁷ M pepstatin A, pH 7.0 with NaOH at roomtemperature) using 1/20th the volume of the 100,000×g supernatant. Theprotein solution was then loaded on a reactive yellow 86 dye ligandcolumn (1 cm×3.5 cm) and the column was washed with 5 column volumes ofrunning buffer. It should be noted that other dye ligand columns can beused in this step of the procedure, when lesser purification factor canbe tolerated. Such dye ligands include by way of illustration and notlimitation reactive blue 4, reactive red 120, reactive blue 2, reactivegreen 5, reactive blue 72, and reactive yellow 3. The D-arabinitoldehydrogenase was eluted by washing the column with 3 column volumes ofrunning buffer supplemented with 1 mM NADH. The eluted protein wasconcentrated using a Centricon 30 microconcentrator device (Amicon) to afinal concentration of at least 1 mg/ml. The D-arabinitol dehydrogenasewas stored at -80° C. The average yield of D-arabinitol dehydrogenasewas 1.0-1.5 mg while purity was greater than 90% as judged by sodiumdodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Table 1summarizes the protein purification results.

                                      TABLE 1                                     __________________________________________________________________________    C. TROPICALIS ATCC 750 PROTEIN PURIFICATION TABLE                                                TOTAL   SPECIFIC                                                              ACTIVITY*                                                                             ACTIVITY                                                                             PURIFIC-                                                 TOTAL (μmoles                                                                            (Activity                                                                            ATION                                              VOLUME                                                                              PROTEIN                                                                             NADH formed                                                                           per mg FACTOR                                                                              YIELD                                 SAMPLE (ml)  (mg)  per min)                                                                              protein)                                                                             (P.F.)                                                                              (%)                                   __________________________________________________________________________    100,000 × g                                                                    55    1661  422.4   0.254  1.00  100                                   Protamine                                                                            60    1888  446.4   0.236  0.93  100                                   SO4                                                                           (NH.sub.4).sub.2 SO.sub.4                                                            3     110.7 225.6   2.038  8.62  53.4                                  Y-86 Column                                                                          0.162 1.2   243.7   203.0  99.62 58.0                                                                    overall                                                                       P.F.                                                                          799.2                                       __________________________________________________________________________     *Activity was determined in 1 ml of 1.5 mM NAD, 50 mM Darabinitol in 50 m     3(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), pH 9.5, 100      mM NaCl, 5 mM MgCl.sub.2 at 25° C. The production of NADH was          monitored by an increase in absorbance at 340 nm over 1 min starting 15       sec after the addition of enzyme.                                        

Example 2

PREPARATION OF MONOCLONAL ANTIBODIES AGAINST CANDIDA TROPICALISD-ARABINITOL DEHYDROGENASE

A. GENERAL METHODS

The standard hybridoma procedures used have been described in detail(Kohler, G.; Milstein, C. Nature 1975, 256, 495-7; Hurrell, J. G. R."Monoclonal Hybridoma Antibodies: Techniques and Applications", CRCPress, 1982, Boca Raton, Fla. 33431).

B. IMMUNIZATION

Balb/C mice (Charles River Laboratories) were immunized with 50 μg ofimmunogen by either intraperitonial or subcutaneous injection. Theimmunogen was prepared by diluting purified D-arabinitol dehydrogenasefrom Candida tropicalis (see Example 1) into Hanks Balanced SalineSolution (HBSS) at a concentration of 500 μg/2.0 ml. The antigen wasthen added to an adjuvant mixture of monophosphoryl lipid A andtrehalose dimycolate in 2% squalene (RIBI Immunochemical Research Inc.)and used for injections. Mice were boosted twice with 50 μg ofimmunogen. A final intravenous injection of 200 μg C. tropicalisD-arabinitol dehydrogenase in HBSS was performed prior to fusion.

C. CELL FUSION

Spleen cells were harvested from the immunized mice and fused with P3X63-AG8.653 myeloma cells (ATCC #CRL 1580) using polyethylene glycol.The cells were resuspended in HAT (0.1 mM hypoxanthine, 0.016 mMthymidine and 0.4 μM aminopterin, Sigma) supplemented media anddistributed into 96-well microtiter plates. Four days later cells werefed by replacing half the HAT supplemented media.

D. HYBRIDOMA SCREENING

Hybridomas were screened for antibodies specific for C. tropicalisD-arabinitol dehydrogenase by a reverse ELISA method. Microtiter EIAplates (Costar #3595) were coated with rabbit anti-mouse IGA, A, M,heavy and light chains (Zymed) at a 1:100 dilution in PBS (0.01M NaHPO₄,0.01M NaH₂ PO₄, 0.015M NaCl, 0.001% NAN₃, adjusted to pH 7.2). 100 μl ofthe above solution was added per well and incubated either at 37° for atleast one hour or at 4° overnight. Any unbound sites on the plate wereblocked with 1% (v/v) normal sheep serum (NSS, Sigma) in PBS (200μl/well). After blotting off the NSS, 50 ul of hybridoma culturesupernatant was added per well. The plates were incubated at 25° for onehour. The plates were then washed four times with ELISA Wash Buffer(0.05% (v/v) Tween-20 in PBS). Excess wash buffer was blotted from theplate and a 2 ug/ml solution of D-arabinitol dehydrogenase in PBT (0.2%(w/v) bovine serum albumin and 0.1% (v/v) Tween-20 in PBS) was added tothe plate at 100 μl/well. The EIA plate was incubated for one hour at25°. The plates were then washed four times with ELISA Wash Buffer andexcess buffer was blotted from the plate. The plates were then developedwith a substrate solution containing 50 mM D-arabinitol (Aldrich) and1.5 mM NAD⁺ in 50 mM CAPSO, 100 mM NaCl, 5 mM MgCl₂ adjusted to pH 9.5.The plates were incubated at 37° for 30 minutes and the NADH product wasmeasured at 340 nm. Alternatively, the production of NADH was coupledwith the reduction of p-iodonitrotetrazolium violet (INT, Sigma) throughthe enzyme diaphorase (Sigma). Diaphorase (0.534 IU/ml, finalconcentration) and INT (0.074 mM, final concentration) were added to theabove reaction mixture. The reaction was incubated as above and theproduction of reduced INT was monitored at 492 nm. Wells with readingsat least three times higher than the background were consideredpositives. Cells from ELISA positive wells were subcloned until stablemonoclonal antibody secretion was achieved. During the subcloning, ELISAscreens were also done substituting C. shehatae D-arabinitoldehydrogenase for the C. tropicalis enzyme.

E. ANTIBODY PRODUCTION IN ASCITES

To scale up monoclonal antibody production in ascites, mice were primedby an intraperitoneal injection of Incomplete Freund's Adjuvant tofacilitate tumor growth 2 to 7 days prior to passage of cells. Cellswere grown up in a T-75 flask, to a final density of about 18×10⁶ cellsin 50 ml, centrifuged, and then resuspended in 2 mL of Dulbecco'sModified Eagle Medium (Gibco) with 10% FBS, 10% NCTC-135 (Gibco), 1 mMoxaloacetic acid (Sigma), 1 mM sodium pyruvate (Gibco), 4 mM L-glutamine(Sigma), 50 ug/mL gentamicin (Gibco), 10 μg/mL insulin (Sigma), 20 mMHepes (Sigma). Each mouse received a 0.5 mL intraperitoneal injection ofapproximately 4-5×10⁶ cells. Ascites tumors usually developed within aweek or two. The ascites fluid containing a high concentration ofantibody was drained from the peritoneal cavity using a hypodermicneedle. The fluid was allowed to clot at room temperature and thencentrifuged at 1500 rpm in a Beckman Model TJ-6 Centrifuge for 30minutes. The antibody containing fluid was poured off and stored at 4°.

Example 3

ELISA BINDING OF MONOCLONAL ANTIBODIES TO DIFFERENT D-ARABINITOLDEHYDROGENASES

Monoclonal antibodies produced against C. tropicalis D-arabinitoldehydrogenase were screened for binding to D-arabinitol specificdehydrogenases purified from C. tropicalis and from C. shehatae by thereverse ELISA method described above. These monoclonal antibodies werealso screened for binding to polyol dehydrogenases that could utilizeother sugar alcohol substrates in addition to D-arabinitol. A C.shehatae polyol dehydrogenase that could utilize D-arabinitol,D-sorbitol, xylitol and D-mannitol was tested. Also tested wasD-arabinitol dehydrogenase from Aerobacter aerogenes, an enzyme thatuses either D-arabinitol or D-mannitol as a substrate.

The D-arabinitol dehydrogenase from C. tropicalis was purified asdescribed in Example 1. The purification of D-arabinitol dehydrogenasefrom C. shehatae was similar to the purification of D-arabinitoldehydrogenase from C. tropicalis with some exceptions. The cellsdisrupted in a Braun MSK Cell Homogenizer were removed from the glassbeads and spun at 30,000×g for 15 minutes to pellet cell wall material.In addition, 60% saturation with ammonium sulfate was used instead of40%, to precipitate the protein. This allowed the precipitation of boththe D-arabinitol dehydrogenase and the polyol dehydrogenase. TheD-arabinitol dehydrogenase was purified over a reactive yellow 86 dyeligand column as described in Example 1. The polyol dehydrogenase fromthis same strain was purified by running the flowthrough from thereactive yellow 86 dye ligand column through a reactive blue 2 dyeligand column (1.0 cm×6.0 cm). The polyol dehydrogenase was then elutedby washing the column with 3 column volumes of running buffersupplemented with 10 mM NAD. D-arabinitol dehydrogenase from Aerobacteraerogenes (lyophilized cells, Type 1, Sigma) was purified as describedin Neuberger, et al., in Biochem. J., 183 (1979) 31-42.

The reverse ELISA screening was as above except a 1:100 dilution ofascites in PBT (100 μL/well) was used instead of culture supernatant. Inaddition, the purified D-arabinitol dehydrogenases from each strain werediluted to 0.2 IU/mL (where an international unit is the amount of NADHproduced/min). As a control, the D-arabinitol dehydrogenase activity wasconfirmed for each activity assay as follows: 10 μL of unboundD-arabinitol was removed from ELISA wells and enzyme activity waschecked with the developing reagents as described above. For a negativeantibody control, Chlamydia trachomatis-immunized mouse sera diluted1:100 in PBT was used.

Results are summarized in Table 2. Each monoclonal antibody was analyzedin duplicate (DUP) with each of the four enzymes. Blanks were run byrepeating the above experiments in the absence of the monoclonalantibodies and blank readings were subtracted from the DUP values. Theaverage of the DUP values is presented in Table 2. The results show thatthe monoclonal antibodies produced against C. tropicalis D-arabinitoldehydrogenase bind the D-arabinitol-specific dehydrogenases from both C.tropicalis and C. shehatae , as evidenced by ELISA readings that areelevated relative to those observed with the Chlamydia antibody. Incontrast, relatively little, if any, binding is seen with the polyoldehydrogenases purified from C. shehatae and from A. aerogenes asevidenced by the fact that these readings are not significantly higherthan those observed with the Chlamydia antibody. These resultsdemonstrate that the monoclonal antibodies bind only D-arabinitolspecific dehydrogenases and do not bind to less specific dehydrogenasesthat utilize either D-arabinitol or other sugar alcohols as substrates.

                  TABLE 2                                                         ______________________________________                                        ELISA BINDING OF POLYOL DEHYDROGENASES                                        TO MONOCLONAL ANTIBODIES SPECIFIC FOR                                         C. TROPICALIS D-ARABINITOL DEHYDROGENASE                                      Antibody                                                                              CTDADH.sup.1                                                                            CSDADH.sup.2                                                                             CSPDH.sup.3                                                                          AADADH.sup.4                              ______________________________________                                        3D6     0.201     0.146      0.009  0.004                                     5E11    0.237     0.169      0.008  0.004                                     1B9     0.171     0.049      0.007  0.003                                     5F3     0.186     0.079      0.006  0.002                                     6B3     0.265     0.060      0.004  -0.002                                    1H4     0.337     0.043      -0.002 -0.001                                    chlamydia                                                                             0.007     0.005      0.003  0.010                                     ______________________________________                                         .sup.1) CTDADH = C. tropicalis Darabinitol dehydrogenase                      .sup.2) CSPADH = C. shehatae Darabinitol dehydrogenase                        .sup.3) CSPDH = C. shehatae polyol dehydrogenase                              .sup.4) AADADH = A. aerogenes Darabinitol dehydrogenase                  

Example 4

A FLUOROGENIC ASSAY FOR D-ARABINITOL IN HUMAN SERUM

Fluorogenic Assay Protocol

Human serum was diluted 1:3 (v/v) with 10 mmol/L citrate (pH 4.0),placed in a boiling water bath for 10 min, and then centrifuged at10,000×g for 10 min to remove precipitated material. The supernatant wasused as the sample. Reaction mixtures (vol.=0.4 mL) contained 0.3 mL ofsample, 1 mmol/L NAD⁺, 4 mmol/L Mg²⁺, 0.35 IU C. tropicalis D-arabinitoldehydrogenase prepared as described in Example 1, and 0.1 mol/L Tris (pH9.5). Incubation was for 15 min at room temperature to promote theD-arabinitol dehydrogenase-catalyzed oxidation of D-arabinitol withconcomitant reduction of NAD⁺ to NADH. This initial incubation stage wasterminated by reducing the pH of the reaction mixture to 5.8 with 0.03mL 1.0 mol/L citrate (pH 3.6). Resazurin (12.5 μmol/L) and diaphorase(0.15 IU) were then added, and the sample was reincubated at roomtemperature to promote the diaphorase-catalyzed utilization of NADH(which had accumulated in the initial incubation stage) for thereduction of resazurin to resorufin. Formation of the latter wasmonitored fluorometrically (Excitation=560 nm, Emission=580 nm) using aPerkin-Elmer fluorescence spectrophotometer, Model #650-40, and wascomplete within 30 sec.

Calibration Curve for the Fluorogenic Assay

Results from a series of assays of a pool of normal human serum intowhich varying amounts of D-arabinitol had been added are summarized inTable 3. A plot of fluorescence emission at 580 nm (Y-axis) vs. amountof D-arabinitol added (X-axis) yields the calibration curve for theassay. The absolute value of the x-intercept of this plot is a measureof the endogenous level of D-arabinitol present in the serum pool. In 22replicate experiments, the calibration curves were linear (mean r²=0.998) and the observed D-arabinitol concentrations in supplementedsamples differed from the expected amounts (y-intercept+amount added) byno more than 0.6 μmol/L or 13.2%.

The results of quadruplicate analyses of a pool of normal human seruminto which 0, 2, 7 and 14 μmol/L D-arabinitol had been added areillustrated in Table 4. The mean measured D-arabinitol concentrationswere within either 0.58 μmol/L or 6.9% of the expected amounts (amountadded plus measured D-arabinitol concentration of the unspiked pool),and the standard deviations were no more than 0.60 μmol/L or 5.9%.

Accuracy of the Fluorogenic D-Arabinitol Assay

The results of analyses of several individual human sera into whichvarious amounts of D-arabinitol had been added are illustrated in Table5. For each serum, the D-arabinitol concentration determined in thesupplemented sample was divided by the sum of the concentrationdetermined in the unsupplemented sample plus the amount contained in thesupplement to yield a measure of the accuracy of the assay. In allinstances, measured D-arabinitol concentrations were within 16% of theexpected amounts. The mean ±SD endogenous D-arabinitol concentration inthe 11 normal adult sera was found to be 1.86±0.36 μmol/L.

Specificity of the Fluorogenic Assay

A series of sugars and sugar alcohols normally present in human serumwere assessed for reactivity in the fluorometric assay. The results aresummarized in Table 6. Of the compounds examined, significant reactivityrelative to that observed with an equimolar concentration ofD-arabinitol was found only with xylitol (3.3%). D-glucose atconcentrations as great as 5.0 mmol/L was not utilized as a substrate.

                  TABLE 3                                                         ______________________________________                                        CALIBRATION CURVE FOR                                                         THE FLUOROGENIC D-ARABINITOL ASSAY                                            D-Arabinitol Added                                                                          Fluorescence Emission, 580 nm                                   (μmol/L)   (Units)                                                         ______________________________________                                        0             19.6                                                            4             50.5                                                            8             78.7                                                            12            111                                                             16            141                                                             ______________________________________                                         y = 19.500 + 7.5825x                                                          r.sup.2 = 1.000                                                          

                  TABLE 4                                                         ______________________________________                                        ACCURACY AND PRECISION OF SERUM D-                                            ARABINITOL DETERMINED WITH THE FLUOROGENIC                                    ASSAY                                                                         D-Arabinitol                                                                           Observed.sup.a                                                       Added    [D-Arabinitol]          Observed/                                    (μmol/L)                                                                            (μmol/L)    CV (%)   Expected.sup.b                               ______________________________________                                        0         1.45 ± 0.086                                                                             5.9      --                                           2         3.43 ± 0.096                                                                             2.8      0.99                                         7        7.83 ± 0.37 4.7      0.93                                         14       15.4 ± 0.60 3.9      1.00                                         ______________________________________                                         .sup.a Mean ± standard deviation of quadruplicate analyses.                .sup.b Observed mean [Darabinitol]/(Darabinitol added + 1.45 μmol/L). 

                  TABLE 5                                                         ______________________________________                                        ACCURACY OF THE FLUOROGENIC D-ARABINITOL                                      ASSAY IN SEVERAL NORMAL HUMAN SERA                                                         Observed                                                                      [D-Arabinitol]                                                                (μmol/L)                                                              D-Arabinitol                                                                             Unsupple-  Supple-                                         Human   Added      mented     mented Observed/                                Serum No.                                                                             (μmol/L)                                                                              Sample     Sample Expected.sup.a                           ______________________________________                                        1       2          1.76       3.94   1.05                                     2       1.5        1.94       3.24   0.94                                     3       3          1.96       4.56   0.92                                     4       10         1.96       10.7   0.89                                     5       75         1.75       85.7   1.12                                     6       7          2.04       8.3    0.92                                     7       8          1.85       10.0   1.02                                     8       40         1.75       48.0   1.15                                     9       1          2.55       3.57   1.01                                     10      8          1.57       9.91   1.04                                     11      2          1.33       2.80   0.84                                     11      5          1.33       5.48   0.87                                     11      10         1.33       9.99   0.88                                     11      45         1.33       44.7   0.96                                     ______________________________________                                         .sup.a Observed [Darabinitol] in supplemented sample/(Darabinitol added +     observed [Darabinitol] in unsupplemented sample).                        

                  TABLE 6                                                         ______________________________________                                        SPECIFICITY OF THE FLUOROGENIC D-ARABINITOL                                   ASSAY                                                                                                   Relative                                            Compound      Concentration                                                                             Reactivity                                          Added         (μmol/L) (%)                                                 ______________________________________                                        D-arabinitol  5           100                                                 L-arabinitol  5           0                                                   ribitol       5           0                                                   xylitol       5           3.3                                                 D-sorbitol    5           0.1                                                 D-mannitol    5           0                                                   erythritol    5           0                                                   threitol      5           0.1                                                 galactitol    5           0.6                                                 D-fructose    500         0                                                   D-galactose   500         0.9                                                 D-mannose     500         0.3                                                 D-glucose     5000        0                                                   ______________________________________                                    

Assays were performed as described in Example 4 except the samplecontained, instead of the boiled serum supernatant, the indicatedcompound in PBS/10 mmol/L citrate, pH 4.0 (1:3,v/v).

Example 5

A RADIOISOTOPIC EXCHANGE ASSAY FOR D-ARABINITOL IN HUMAN SERUM

Tritium Exchange Assay Protocol

Assay incubation solutions (vol.=0.1 mL) contained 0.05 mL ultrafilteredhuman serum, 0.2 mmol/L unlabeled NAD⁺, 0.015 mmol/L (0.62 Ci/mmol)[4(S)-³ H]NADH, 2 mg/mL BSA, 0.2 mmol/L DTT, 4 mmol/L Mg²⁺, 40 mmol/LTris (pH 9.2), and 0.35 IU C. tropicalis D-arabinitol dehydrogenase.Incubation was for 2 hr. at room temperature to promote the D-arabinitoldehydrogenase-catalyzed exchange of tritium from 4(S)-³ H]NADH intoD-arabinitol. After incubation, the sample was diluted with 0.9 mLdistilled water and applied to a 2 mL column of AG 2-X8 (Bio Rad, Cat.No. 731-6247) preequilibrated in the ⁻ OH form. The column was elutedwith 6 mL distilled water and the eluate was collected, diluted 1:1(v/v) with 250 mmol/L NH₄ OAc (pH 8.8), and applied to a 1 mL column ofphenylboronate (Pierce, Cat. No. 20368) preequilibrated with the NH₄ OAcbuffer. The column was washed with 10 mL 250 mmol/L NH₄ OAc (pH 8.8) andsubsequently eluted with 4 mL 0.1 formic acid. The formic acid eluatewas analyzed for tritium by scintillation spectrometry.

Calibration Curve For the Tritium Exchange Assay

Results from a series of assays of a pool of normal human serum intowhich various amounts of D-arabinitol had been added are illustrated inTable 7. A plot of tritium recovered (Y-axis) vs. the amount ofD-arabinitol added (X-axis) yields the calibration curve for the assay.The absolute value of the X-intercept of the curve is a measure of thesum of endogenous D-arabinitol and D-ribulose present in the serum pool.The endogenous serum concentration of D-ribulose can be obtained bycarrying out the assay under similar conditions but in the absence ofNAD⁺ and determining tritium incorporation into D-arabinitol. Thecalibration curve is linear (r² =0.990) and the observed D-arabinitolconcentrations in supplemented samples differed from the expectedamounts (endogenous +amount added) by no more than 0.42 μmol/L or 7.9%.

                  TABLE 7                                                         ______________________________________                                        CALIBRATION CURVE FOR THE TRITIUM EXCHANGE                                    ASSAY                                                                                     D-Arabinitol                                                                             Tritium                                                Assay       Added      Recovered                                              No.         (μmol/L)                                                                              (cpm × 0.001)                                    ______________________________________                                        1           0          25.5                                                   2           2          45.3                                                   3           4          62.8                                                   4           6          72.9                                                   5           8          90.8                                                   ______________________________________                                         y = 27.82 + 7.91x                                                             r.sup.2 = 0.990                                                          

Example 6

AN AUTOMATED CHROMOGENIC ASSAY FOR D-ARABINITOL IN HUMAN SERUM

Automated Chromogenic Assay Protocol Assays were performed on theCOBAS-MIRA (Hoffmann LaRoche). Samples were human sera pretreated byboiling as indicated in the fluorometric assay protocol. In addition toa diluent (1 mol/L glycine, pH 10.5), the following three reagents wereemployed:

1. Enzyme Reagent (10 mmol/L Tris/Acetate (pH 6.0), 100 mmol/L NaCl, 10mmol/L Mg²⁺, 2.33 mmol/L NAD⁺, 20 μg/mL BSA, and 3.5 IU/mL C. tropicalisD-arabinitol dehydrogenase).

2. Coupling Reagent (3.18 mmol/L iodonitrotetrazolium (INT), 66.7 mmol/LEDTA (pH 9.0) and 0.03% Pluronic 25R2).

3. Diaphorase Reagent (60 U/mL C. kluyveri diaphorase (Sigma, Cat. No.02381) in PBS).

With a cycle time of 25 seconds and an assay incubation temperature of30° C., the following assay steps are carried out by the MIRA:

Cycle 1 mix sample (vol.=85+10 μL diluent)+enzyme reagent (vol.=100 μL).

Cycle 37 add coupling reagent (vol.=15+5 μL ddH₂ O)

Cycle 38 read Abs₅₀₀ nm

Cycle 39 add diaphorase reagent (vol.=3+5 μL ddH² O)

Cycle 40 read AbS₅₀₀ nm

The assay result is reported as ΔA=Abs₅₀₀ nm, cycle 40-Abs₅₀₀ nm, cycle38.

Calibration Curve for the Automated Chromogenic Assay

Results from a series of assays of a pool of normal human serum intowhich various amounts of D-arabinitol had been added are illustrated inTable 8. The endogenous level of D-arabinitol in this serum pool waspreviously determined to be 1.0 μmol/L by the enzymatic method of Wongand Brauer (J. Clin. Microbiol. 26, 1670 (1988). A plot of mean ΔAbss₅₀₀nm (Y-axis) vs. total serum D-arabinitol (endogenous +amount added)(X-axis) yields the calibration curve for the assay. The calibrationcurve is linear (r² =1.00) and the observed D-arabinitol concentrationsin supplemented samples differed from the expected amounts(endogenous+amount added) by no more than 0.17 μmol/L or 1.0%.

                  TABLE 8                                                         ______________________________________                                        CALIBRATION CURVE FOR THE                                                     AUTOMATED CHROMOGENIC ASSAY                                                               Serum                                                             Assay       D-Arabinitol                                                                             Mean ΔAbs.sub.500nm                              No.         (μmol/L)                                                                              (× 10.sup.3)                                     ______________________________________                                        1           1.0        9.4                                                    2           6          16.5                                                   3           11         23.65                                                  4           16         31.35                                                  5           21         38.45                                                  6           26         45.45                                                  ______________________________________                                         y = 7.89 + 1.45x                                                              r.sup.2 = 1.00                                                           

The cell lines designated DADH 1-5E11 and DADH 1-3D6, were deposited onJun. 18, 1991 with the American Type Culture Collection (ATCC), locatedat 12301 Parklawn Drive, Rockville, Md. 28852, U.S.A., and received ATCCdesignations HB 10776 and HB 10777, respectively.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes or modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A composition comprising (1) a D-arabinitoldehydrogenase in purified form having the characteristics of aD-arabinitol dehydrogenase obtainable from Candida tropicalis ATCC 750or Candida shehatae said characteristics being a specific activity forcatalysis of the oxidation of D-arabinitol to D-ribulose of at least 50international units/mg and having a specific activity for catalysis ofthe oxidation of D-mannitol of less than 1% of the specific activity forthe catalysis of the oxidation of D-arabinitol; and (2) NAD⁺, saidcomposition being capable of catalyzing the oxidation of D-arabinitol ata rate at least 20-fold faster than it catalyzes the oxidation of sugarsand sugar alcohols present in human serum.
 2. The composition of claim 1wherein the dehydrogenase is obtained from an organism of the genusCandida.
 3. The composition of claim 2 wherein the dehydrogenase isobtained from the organism Candida tropicalis.
 4. The composition ofclaim 2 wherein the dehydrogenase is obtained from the organism Candidashehatae.